There is extensive evidence from laboratory animal studies and human clinical trials that dietary flavonoids derived from fruits and vegetables protect against inflammation in the intestinal tract and also induce health benefits in distal organs. The genesis of the health-promoting effects of individual flavonoids and their mixtures has been linked not only to their direct effects but also to their metabolism by intestinal microbes and to their alteration of intestinal microbial populations. The aryl hydrocarbon receptor (AhR) and its ligands also play a protective anti-inflammatory role in the intestine. The overall hypothesis of this proposal is the anti-inflammatory activities of flavonoids are due, in part, to their structure-dependent activity as AhR ligands and their interactions with the microbiome. Based on exciting new preliminary data showing structure- dependent activity of flavonoids as inducers of Cyp1A1 and IL-22 (a key anti-inflammatory, epithelial regeneration response gene), Aims 1a&b will characterize the structure-dependent effects of flavonoids on AhR signaling using colonic cells and in vitro gut epithelial models. Aim 1c will examine the ability of flavonoids to suppress inflammation by modulating the AhR-IL-22 signaling axis in immune cell populations. Aim 2 will use multi-omic analytics to first identify microbial metabolites of flavonoids using complementary in vitro and in vivo assays (Aim 2a&b), and Aim 2c will use a novel computational approach to associate specific flavonoid metabolites with their source microorganisms. Aim 3a will examine the AhR-mediated effects of flavonoids and their metabolites in both the colonic cell lines and organoid 3d cultures. In addition, Aim 3b will investigate the in vivo ability of flavonoid extracts to modulate the AhR mediated regenerative, antimicrobial response to chemical or genetically induced mucosal injury in GI- specific AhR KO mice. By contrasting intestine epithelium-specific AhR KO with wild type control mice, the contribution of the epithelial vs stromal (containing infiltrating immune cells) AhR mediated regenerative response to mucosal injury will be definitively determined.